1. Field of the Invention
This invention relates to the field of biochemical analysis by mass spectrometry. More specifically this invention relates to the optimization of biological sample processing for mass spectrometric analysis; in particular for biological agent sample preparation for mass spectrometric analysis using matrix assisted laser desorption/ionization (MALDI) ion sources or atmospheric pressure MALDI ion sources for generation of ions.
2. Background of the Invention
Mass spectrometry has been used to analyze microorganisms. Biomarkers, cellular material specific to these microorganisms, such as proteins and peptides have been found to be characteristic of a given organism such as bacterial spores or viruses. Toxins are proteins and are analyzed similarly.
MALDI mass spectrometry (MS) has been used for the analysis of biomolecules with large molecular weight. MALDI techniques can detect molecular ions with masses greater than 100,000 Da. MALDI is suitable for analyzing complex mixtures (without prior mixture separation) and for this reason it is considered as a suitable technique for peptide/protein characterization.
Organism specific biomarkers (usually proteins or peptides) have been identified using various combinations of sample processing techniques and identified by mass spectrometry. In order for mass spectrometry to produce the signals of these biomarkers, the microorganisms should be purified from the rest of the cellular material that is present. This is usually followed by sample concentration. Chromatography is commonly used for these purposes with the separation usually taking more than 15-30 minutes. After separation, the microorganism samples are typically treated with enzymes to provide biomarker peptides that can be analyzed by mass spectrometry or tandem mass spectrometry (or MS/MS).
The resultant tandem mass spectral data are typically provided to a proteomic database search, and the organisms are identified from the particular protein returned by the database. Proteomic databases are utilized for comparison to and identification of proteins based on comparison of peptide sequence information obtained in the MS/MS analysis with those available in the proteome/genome database and subsequently provide the identification of the protein and then the organism.
In the case of MALDI or atmospheric pressure (AP) MALDI, a separation step is not necessary, and biological samples are processed on a sample holder (e.g., a probe or a MALDI target plate). The biological samples may be selectively dissolved, or in the case of whole cells the cells may be lysed on the plate releasing the biomarkers. This sample processing, combined with MALDI analysis can provide protein signatures which can be matched to the genome of a specific biological entity or bacteria. This permits identification of microorganisms, which can also be used as biomarkers of common functional diseases and disorders. Sequence determination of such biomarkers can be achieved with high throughput.
The following articles related to sample preparation, processing, and analysis have been reported in the scientific literature, all of which are incorporated herein in entirety by reference:    1. Doroshenko, V. M.; Laiko, V. V.; Taranenko, N. I.; Berkout, V. D.; Lee, H. S. (2002), “Recent developments in atmospheric pressure MALDI mass spectrometry” Int. J. Mass Spectrom. 221: 39-58.    2. Eng, J. K., A. L. McCormack, et al. (1994). “An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.” Journal of the American Society for Mass Spectrometry 5(11): 976-989.    3. Fenselau, C. and P. A. Demirev (2001). “Characterization of intact microorganisms by MALDI mass spectrometry.” Mass Spectrom. Rev. 20(4): 157-171.    4. Harris, W. A. and J. P. Reilly (2002). “On-Probe Digestion of Bacterial Proteins for MALDI-MS” Anal. Chem. 74(17): 4410-4416.    5. Hooker, J. M., E. W. Kovacs, and M. B. Francis, Interior surface modification of bacteriophage MS2. J Am Chem Soc, 2004. 126(12): p. 3718-9.    6. Karas, M. and F. Hillenkamp (1988). “Laser desorption ionization of proteins with molecular masses exceeding 10000 Daltons.” Anal. Chem. 60(20): 2299-2301.    7. Krishnamurthy, T. and P. L. Ross (1996). “Rapid identification of bacteria by direct matrix-assisted laser desorption/ionization mass spectrometric analysis of whole cells.” Rapid Commun. Mass Spectrom. 10: 1992-1996.    8. Krutchinsky, A. N., M. Kalkum, et al. (2001). “Automatic Identification of Proteins with a MALDI-Quadrupole Ion Trap Mass Spectrometer.” Anal. Chem. 73: 5066-5077.    9. Perkins, D. N., D. J. Pappin, et al. (1999). “Probability-based protein identification by searching sequence databases using mass spectrometry data.” Electrophoresis 20(18): 3551-67.    10. Pribil P A, Patton E, Black G, Doroshenko V, Fenselau C. (2005), “Rapid characterization of Bacillus spores targeting species-unique peptides produced with an atmospheric pressure matrix-assisted laser desorption/ionization source.” J Mass Spectrom. 40(4): 464-474.    11. Strauss, J. H., Jr. and R. L. Sinsheimer, Purification and properties of bacteriophage MS2 and of its ribonucleic acid. J Mol Biol, 1963. 7: p. 43-54.    12. Tanaka, K., H. Waki, et al. (1988). “Protein and polymer analyses up to m/z 100,000 by laser ionization time-of-flight mass spectrometry.” Rapid Commun. Mass Spectrom. 2: 151-153.    13. Warscheid, B., and Fenselau, C. (2003). “Characterization of Bacillus Spore Species and Their Mixtures Using Postsource Decay with a Curved-Field Refl ectron,” Anal. Chem. 75(20): 5618-5627.
Despite this work, improved sample preparation and processing techniques are still being pursued.